AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

stable for only 90 days stored in the freezer at -70 o C. f.) Pteroyltri-y-glutamate (FA-glu 3

) is stable for 12 months stored in the freezer at -70 o C.

Preparation of Stock standards of different folate compounds

Table 2: Preparation of Stock standard solns. (examples) of different folate compounds

Folate compd #

Final Vol* (mL)

Concen (mcg/mL)

Standard

Mass (g)

Shircks (6S)-5-Formyl-5,6,7,8- tetrahydrofolic acid

1

0.00607

10

607

2

10-Formylfolic acid

0.00597

10

597

Shircks (6R,S)-5-methyl-5,6,7,8- etrahydrofolic acid

3

0.00584

10

584

4

10-methylfolic acid

0.00529

10

529

Shircks (6R,S)-5,10-methenyl- 5,6,7,8-tetrahydrofolic acid

5

0.00587

10

587

6**

pteroyltri-y-L-glutamic acid

0.00911

10

911

Shircks (6S)-5,6,7,8-tetrahydrofolic acid

7

0.00542

10

542

8

Shircks Folic acid

0.00555

10

555

* make up volume to 10 mL with stock solution solvent. **used only for checking conjugase activity in rat plasma

5). Folate Internal std.Stock solutions:

a. 13 C-Folic Acid Internal Standard Stock soln. (2000 ug/mL) - The labeled Folic acid, 13 C-Folic Acid Millipore / Merck CH2QC-900824. The chemical is usually bought in 20 mg amount. The entire 20 mg amount of labeled folic acid is dissolved in 10 mL of folate stock std. solvent. Dissolve the chemical completely with the aid of vortex and brief (15 second) sonication. The solution is anticipated to be stable for up to 5 months at -70 o C. Stability to be checked during and after this period before use. b. 13 C5-(6S)-5-Methyl-5,6,7,8-Tetrahydrofolate Internal Standard Stock soln. (2000 ug/mL) : The labeled methyl-THF , 13 C5-(6S)-5-Methyl-5,6,7,8- Tetrahydrofolate (Millipore / Merck CH2QA-900827). The chemical is usually bought in 20 mg amount. The entire 20 mg amount of labeled methyl-THF is dissolved in 10 mL of folate stock std. solvent. Dissolve the chemical completely with the aid of vortex and brief (15 second) sonication. The solution is anticipated to be stable for up to 3 months at -70 o C. Stability to be checked during and after this period before use

6. Folate Intermediate Standard Solution (20ug/mL):

7